We are interested in the understanding protein-ligand interactions and the microevolutionary processes that permit and promote changes at the protein-ligand interface. To explore these issues, we are studying the Phd repressor/antitoxin and selected homologs. Phd is a small 73 amino acid protein with multiple macromolecular ligands. Phd dimerizes, binds to operator DNA, binds to the Doc toxin, and engages in an additional contact with Doc that mediates the cooperative interactions between adjacent Phd dimers and thus enhance repression. All four of these protein-ligand interactions contribute to the specificity and affinity of the repressive complex. The specific aims of the current proposals are: 1) To determine how the spacing and sequence of the palindromic sites in the operator affects the formation of the repressive complex. 2) To develop quantitative in vitro assays for all four interactions. 3) To determine the role of the two structurally identified Phd-Doc interactions on a) toxicity, b) neutralization of the toxin and c) corepression. The long-term objective of this research is to understand protein-ligand interactions well enough to recognize ligand binding domains, match proteins to their preferred ligands, design proteins to bind specific ligands and design ligands to bind specific proteins. A superior understanding of protein-ligand interactions will assist in the rational design of drugs (ligands) that interact with specific protein targets (agents of disease). PUBLIC HEALTH RELEVANCE: The proposed research will contribute to the understanding of protein-ligand interactions and a superior understanding of protein-ligand interactions will enable us to design ligands (drugs) that bind specific proteins (viral or microbial or human) that produce human disease. The proposed research will also contribute to the understanding of plasmid addiction elements. These self-selecting sib-killing plasmid addiction elements may have diverse applications in the attenuation of bacteria for vaccine production, in enhancing and prolonging the action of antibiotics and in the modification or elimination of plasmid-borne virulence determinants.